Screening and Optimization of IAA Production by PGPR isolated from Rhizosphere of a Pterocarpus marsupium Roxb. and their Effect on Plant Growth

Introduction

The rhizosphere is an intimate area of soil near the root system of the plant¹. The rhizosphere consists of a micro-ecosystem that involves the root system of a plant, microorganisms, nematodes, etc.². The interaction between microorganisms and plants is special and beneficial to each other. Microbial components consist of bacteria and fungi, having multifarious plant growth-promoting potential such as nutrient acquisition (nitrogen fixation, phosphate/ zinc/ potassium solubilization), Siderophore production, ACC deaminase production, plant growth-promoting hormones(Auxins, Gibberellins)etc.³. IAA is an important auxin that regulates plant growth and development, and also act an important signaling molecule. Ortiz-Castro R et al.(2012)⁴ and Raheem et al.(2018)⁵ have reported that IAA-producing bacteria viz, Bacillus amyloliquifacience significantly increased the length of the wheat plant and B. muralis D-5 and Enterobacter aerogenes S-10 had a positive impact on spike length and seed weight under drought stress. IAA-producing plant growth-promoting microorganisms (PGPM) include Pseudomonas, Bacillus, Azotobacter, Acinetobacter, Rhizobium, Bradyrhizobium, Burkholderia, Candida tropicalis, Ustilago esculanta etc.^6,7. Cultural conditions for IAA production is widely studied, major factor affecting IAA production are pH, temperature and tryptophan concentration, nitrogen source, days of incubation, etc.⁸.

Results of optimization  studies in Pseudomonas putida UB1 revealed that  addition of L-tryptophan at concentration of  0.2 mg/mL at pH 7.5  and 96 hrs of incubation produced maximum IAA⁹. Rhizobium spp. produced the highest IAA (166 μg/mL) at a temperature of 36°C, pH of 6.5, an incubation time of 24hrs. and respectively tryptophan and NaCl concentrations of 1 g/L and 0.1 g/L¹⁰.

 Wheat is one of the top three grains consumed worldwide. India is the world’s second largest producer of wheat, although rising production per capita is quite low in contrast to current demand^11,12, use of chemical fertiliser is significantly enhances output, but its impact on ecosystem and soil health cannot be neglected¹³. Biofertilizers and compost application can reduce reliance on chemical fertilizers while also ensuring sustainable agriculture. According to reports, using compost, sludge, and Azotobacter has increased wheat production and nutritional content when compared to artificial fertiliser¹⁴. Microbial community composition of rhizosphere is unique to plant and is affected by various biotic and abiotic factors such as plant genotype, age of the plant and environmental factors, various anthropogenic activities etc., respectively.

Research on Arabidopsis thaliana has revealed that, while grown under identical conditions, the seven cultivars exhibited distinct rhizodeposit compositions as well as the development of a genotype-specific rhizobacterial community¹⁵. Moreover, the architecture of the root system is influenced by the rhizodeposit’s composition¹⁶. Because native plants are confined to a specific region, their rhizosphere microflora must be distinct and may have agricultural significance, though such studies are meagre. Pterocarpus marsupium Roxb (Family: Fabaceae)  is a large, indigenous, deciduous tree from Northern Western Ghats. Because of their great medicinal value, all the parts of this species have been used in homoeopathic, ayurvedic, and unani medical systems. Its rhizosphere could provide promising PGPR that is significant to agriculture.

The current study aims to isolate and screen IAA-producing PGPR from rhizosphere of  Pterocarpus marsupium Roxb’s. Optimization study for IAA production with selected isolates for parameters viz, pH, temperature, tryptophan concentration, and incubation period and their effect on root development of wheat plant was investigated.

Materials and Methods

Soil sample  Rhizosphere soil of Pterocarpus marsupium Roxb  (Location: Paud Ghat, Dist- Pune, Maharashtra, India) was collected from 20- 30 cm depth from the ground, soil firmly attached to the root was taken along with root hairs in a sterile container and was further processed in laboratory conditions.

Isolation and screening of bacteria from rhizospheric soil

10 gm of rhizosphere soil was inoculated in the sterile nutrient broth and incubated at 28^oC for 48hrs.  Serially diluted sample was plated on a sterile nutrient agar plate and incubated at 28^oC for 3-4 days; colony characteristics of all obtained isolates were noted. All isolates were purified and preserved on nutrient agar slants and stored at 4^oC for further use.

 Quantitative screening of IAA producing bacteria

 For quantitative screening of IAA production by bacterial isolates, Salkowsky reagent(0.5% FeCl3 in 70% perchloric acid)⁸ was used.  The sterile Luria broth with pH 7 and 0.1mg/mL of tryptophan was inoculated with   the test bacterial cultures, and incubated at 28^oC for 7 days. The cultures were then centrifuged, 1ml of supernatant was added with 2ml of Salkwasky reagent and incubated at room temperature for 30 minutes in the dark reaction developed pink colour, intensity of colour was measured calorimetrically at 430 nm against  blank. The standard curve of IAA, was prepared using the standard concentrations of IAA (Sigma Aldrich) in Luria broth, which was further used to calculate the concentration of IAA from test samples ^6,17.

Detection of IAA by Thin layer chromatography (TLC)

The standard IAA (0.1 mg/mL in methanol) and crude IAA (produced in the previous experiment) were spotted on a 15 cm x 10 cm TLC plate (Silica gel Gf 254, thickness 0.25 mm) and separated in solvent system conatining n-butanol, ethyl acetate, ethanol, and water in the ratio of 3:5:1:118. The plate was then developed with Ehmen reagent( 98 mL of 35% HClO₄ mixed with 2 mL of 0.5M FeCl₃)¹⁵. The plate was heated at 90^oC for 3-4 minutes for visualization of spots. R_f values of test samples and standard IAA were compared and those which coincide with standard IAA were identified^16,17.

Optimization of culture condition for maximum IAA production for selected isolates

The effect of pH, incubation temperature, incubation period, and tryptophan concentration on IAA production was studied using classical method ^6,8,10,18,19 for the five top IAA-producing isolates, the procedures for the same are listed below:

pH

To study effect of pH on IAA production, Luria broth at pH 5, 6, 7, and 8 was supplemented with 0.1 mg/mL of tryptophan, inoculated individually with bacterial isolates, and incubated for 4 days at 28 ^oC. Concentration of IAA  in test samples were determined by using Salkowsky reagent.

Incubation temperature

Effect of temperature on IAA production was studied by inoculating test bacterial samples in Luria broth of pH 7 supplemented with 0.1mg/mL of tryptophan and incubated for 4 days at temperatures 25, 30, 35, 40^oC with continuous shaking at 100 rpm. IAA from test samples was determined by using Salkowsky reagent.

Incubation period

Each test bacterial isolates was separately inoculated in Luria broth of pH 7 containing 0.1mg/mL tryptophan and incubated at 28^oC in shaker incubator at 100 rpm. Sample from each flask was collected at 24hrs, 48hrs, 72hr, and 96hrs of incubation and cell free broth was assessed for IAA using Salkowsky reagent.

Tryptophan concentration

Effect of tryptophan concentration on IAA production was determined by inoculating Luria broth of pH 7 supplemented with L-tryptophan at concentration of 0.05, 0.1, 0.5, and 1mg/mL with test bacterial isolates separately and incubated for 4 days at 28^oC, IAA from all tubes were determined by using Salkowsky reagent.

Effect of selected PGPR on growth of Wheat

Effect IAA producing PGPR was studied on growth and root architecture of Wheat (Triticum aestivum) MACS 6222 variety. MACS 6222 is high yielding, rust resistant and commonly grown variety in India especially Maharashtra. Wheat seeds were procured from MACS-Agharkar Research Institute (ARI), Hol farm, Dist-Pune Maharashtra India. Seeds were washed with few drops of Tween 20 and water thoroughly to remove detergent completely and further surface sterilized with 0.1% HgCl₂ for 1 minute, and washed with sterile distilled water five times. Surface sterilized seeds were treated with each test bacterial inoculum containing 10⁸cells/mL overnight, coated seed were dried and 20 seeds of each treatment were placed in a petri plate on moist paper towel. All the petri plates were incubated in at 25^oC for 10 days. Watering was done as per requirement. The physiological parameters viz., germination time and germination percentage, plant height, fresh biomass, root morphology were noted. Experiment was carried out in triplicate.

Statistical analysis

 The results of optimization of IAA are presented as means of three replicates ± standard deviation. Significance of PGPR treatment on plant growth and root development were statistically analysed using one-way analysis of variance (ANOVA) and  means were compared using Dunnet multiple comparison test at   p = 0.05 in Minitab18 statistical software .The graphs were plotted using MS excel and Minitab18 software. 

Results and Discussion            

A total of twenty four isolates were obtained from the rhizosphere of Pterocarpus marsupium Roxb., colony characteristics are mentioned in Table1. Isolates were further screened for quantitative IAA production using the Salkowsky reagent. All of the isolates displayed pink coloration, indicating that they are all capable of producing IAA (Fig1). Five bacterial isolates viz; Et1, Rp1, Rp5, Rp6, Rp9 with higher potential for IAA production (viz 50, 61,70, 76, 70 μg/mL respectively) were employed in further studies. ( Table2).

Table 1: Colony characteristics of bacterial isolates from rhizosphere of  Pterocarpus marsupium Roxb. 

Isolate	Size( mm)	shape	Colour	Margin	Opacity	Consistency	elevation
Et1	2	Circular	Off white	Entire	Opaque	Soft	Flat
Et2	2	Circular	Cream	Entire	Opaque	Soft	Flat
Et3	6	Circular	Off white	Irregular	Opaque	Soft	Convex
Et4	3	Circular	White	Entire	Opaque	Soft	Umbonate
Et5	4	Circular	White	Irregular	Translucent	Soft	Umbonate
Et6	5	Circular	Creamy yellow	Entire	Translucent	Sticky	Flat
Et7	3	Circular	Cream	Irregular	Opaque	Soft	Convex
Et8	2	Circular	Light yellow	Entire	Opaque	Soft	Slightly raised
Et9	1	Circular	Cream	Entire	Translucent	Soft	Flat
Et10	3	Circular	Off white	Undulate	Opaque	Soft	Flat
Et11	4	Circular	Yellow	Entire	Opaque	Soft	Convex
Et12	2	Circular	Cream	Entire	Opaque	Soft	Erose
RP1	4	Circular	Cream	Entire	Translucent	Soft	Flat
RP2	3	Circular	Off white	Entire	Translucent	Sticky	Flat
RP3	3	Circular	Shiny white	Entire	Translucent	Mucoid	Flat
RP4	4	Circular	Cream	Irregular	Opaque	Sticky	Convex
RP5	3	Circular	White	Entire	Opaque	Soft	Crateriform
RP6	1	Circular	White	Entire	Opaque	Soft	Raised
RP7	1	Circular	Cream	Entire	Opaque	Soft	Flat
RP8	3	Circular	White	Entire	Opaque	Soft	Flat
RP9	3	Circular	Off White glossy	Entire	Opaque	Mucoid	Convex
RP10	2	Circular	Off white	Entire	Translucent	Soft	Flat
Ed1	5	Circular	Cream	Entire	Opaque	Soft	Raised
PT1	3	Circular	Off white	Entire	Opaque	Hard	Convex

Figure 1: IAA production by bacterial isolates obtained from rhizosphere of Pterocarpus marsupium Roxb, pink colour indicates presence of IAA. Click here to view Figure

Table 2: Quantitative IAA production by bacterial isolates obtained from rhizosphere of Pterocarpus marsupium Roxb. using Salkowsky reagent.

Isolate	(IAAμg/ml)	Isolate	IAA(μg/mL)
Et1	50	Rp1	61
Et2	27	Rp2	26
Et3	24	Rp3	42
Et4	24	Rp4	37
Et5	28	Rp5	70
Et6	18	Rp6	76
Et7	24	Rp7	35
Et8	23	Rp8	32
Et9	25	Rp9	70
Et10	25	Rp10	29
Et11	16	Ed1	31
Et12	14	PT1	22

 Optimization studies on IAA production

Bacteria produce IAA by tryptophan-dependent and tryptophan-independent pathways, however majority of bacteria synthesize high amount of  IAA in presence of L- tryptophan, as tryptophan is a precursor of IAA^18,20. The L-tryptophan content, temperature, incubation time, and medium pH were the factors that optimized for IAA production by top five producers using the classical method (One Factor at a Time). The current study’s findings show that tryptophan supplementation in media raises IAA production. Mohite( 2013)⁶ and Duca et al.( 2014)²¹ support tryptophan’s beneficial effects on IAA production. Maximum IAA was produced at 0.1mg/mL of tryptophan by all five isolates i.e 54, 62, 73, 78, 72 mg/mL, respectively. Tryptophan concentrations between 0.05 and 0.1 mg/ml produced almost equal amount of IAA, although, IAA production is reduced at higher tryptophan concentrations. These results validate the findings of Shokri & Emtiazi(2010)¹⁹, who found that higher tryptophan concentrations have a negative impact on IAA production. However, Bharucha et al.(2013)⁹ have also reported maximum IAA production by Pseudomonas putida UB1 at 0.2% tryptophan. (Fig 2a)

 Metabolic activities of microorganisms are highly affected by physiological factors such as pH, temperature, macro, and micronutrient sources and their concentration, etc. Every organism has a cardinal range of the above factors for growth. IAA production is also affected by factors viz., temperature, pH, tryptophan concentration, nitrogen source, NaCl, incubation period etc.^8,23,24.

There is a diverse report on the temperature requirement and incubation period for maximum IAA production. In the current investigation four isolates (Et1, Rp1, Rp5, Rp9) have produced maximum IAA i.e 50, 61, 71,71μg/mL respectively at 30^oC and decreased further with increase in temperature; whereas RP6 produced maximum IAA (84.12 μg/mL ) at 35^oC  (Fig 2b). All the isolates produced highest IAA at 96 hrs. of incubation(Fig.2c). Results are in support of Patten&Glick(2002)²⁵, they have obtained maximum IAA production by Pseudomonas putida. GR12–2 in 96 hrs and  beyond 96hrs IAA concentration was found to be decreased; B. siamensis also produces maximum IAA at 35^oC in 96 hrs²⁶. Shokri and Emtiaz(2010)²² have reported 30^oC and 72 hrs  as an optimum temperature and incubation period for Paenibacillus, and Rhizobium strains. IAA production was found to be increased with the incubation period as IAA production occurs at the stationary phase of growth¹⁰. Rhodopseudomonas palustris  produced maximum IAA (80.77± 2.13 μg/mL) at 35^oC in 48hrs²⁷.

Figure 2: Effect of different physiological parameter on IAA production (2a- Tryptophan concentration 2b- Incubation temperature 2c -Incubation period  2d- pH). Click here to view Figure

pH significantly affects the synthesis of IAA. A wide range of pH values have been found to be ideal for the synthesis of IAA²⁷. In our investigation it was found that the synthesis of IAA is adversely affected by an acidic pH.  At pH 7, all five isolates produced the highest amount of IAA, which ranges from 53 to 74 μg/mL. It was shown that Rp1 could produce almost the same amount of IAA (59.2 & 61 μg/mL) at pH values of 6 and 7, respectively; Rp6 produced the maximum amount of IAA (74 and 73 μg/mL, pH values of 7 and 8), and Rp9 produced nearly the same amount of IAA at pH values of 6 (68.71 μg/mL), 7 (71), and 8 (71.67g/mL) (Fig 2d). Mohite (2013)⁸ has also examined this fact, that distinct isolates generated maximum IAA at varying pH values. Lebrazi, Niehaus, et al. (2020)¹⁶ report that Rhizobium produces highest levels of IAA at pH 6.5, whereas Bharucha et al(2013)⁹ reported that Pseudomonas putida UB19 has a pH of 7.5.

Detection of IAA by thin-layer chromatography (TLC)

The developed chromatogram showed pink colour spots with all bacterial samples and standard IAA. The bacterial sample showed two spots each one with an R_f value of 0.91 which matches with standard IAA while another spot is of R_f value 0.23 which remains to be identified (Fig 3). Our results of TLC of IAA are consistent with Shokri & Emtiazi(2010)²² and Kang et al(2019)²⁸ studies. On TLC plates, one more component that has not yet been named was found in addition to IAA.

Figure 3: Detection of IAA by Thin layer Chromatography (TLC). Click here to view Figure

(Lane1- Std IAA, Lane 2- Et1, Lane 3- Rp1, Lane4- Rp5. Lane5- Rp6, Lane6- Rp9 with  R_f = 0.94 )

Effect of PGPR on Wheat growth and root system development

Beneficial effect of bacterial isolates Et1, Rp1, Rp5, Rp6 and Rp9 were evaluated on wheat growth. The statistical analysis of variance showed that all the five isolates significantly increase the plant height in comparison with untreated control as P = 0.00 at 0.05 % level of significance. Dunnets’s Simultaneous comparison at 95% confidence intervals also suggests means of height and biomass of all treatment are significantly different from untreated control mean (Fig 4a,4b,).  Similarly analysis of variance of plant biomass give value of P =0.00 at 0.05% level of significance, Dunnet simultaneous comparison indicates that there is a significant difference between mean of untreated control and ET1, RP5 and RP6 but treatment RP1 and RP9 does not have significant difference in mean biomass with untreated control(Fig 4c and 4d).

Figure 4a: Effect of PGPR treatment on height (mean± SD) of wheat seedlings along with untreated control. Click here to view Figure

Figure 4b: Comparison of height of  wheat seedling with Individual treatment and untreated control by Dunnett simultaneous comparison at 95% CI Click here to view Figure

Figure 4c: Effect of PGPR treatment on biomass (mean ± SD) of wheat seedlings  Click here to view Figure

Figure 4d: Comparison of biomass wheat seedling with individual treatment and untreated control by Dunnett simultaneous comparison at 95% CI Click here to view Figure

Rp6 appears to be most promising among all five as it causes approximately 50% increase in plant height and biomass followed by Rp5 (40%) and Et1 (30%) (Table3,Fig5a), these results are in agreement with studies performed by Dahmani et al.(2020)²⁹, in which plant growth promoting  Bacillus megaterium strain (RmBm31) increases root biomass and positively develops root architecture of  Arabidopsis thaliana. Similar to this, Ashrafuzzaman et al.(2009)³⁰ showed that rice rhizosphere isolates generated IAA and impacted plant height and root length of rice seedlings. 

Table 3: Effect of bioinoculation on height and biomass of wheat plant ( 10 days seedling)

Treatment	Avg height(m)	Biomass(g)
Et1	0.16 ±0.01	0.14±0.018
Rp1	0.12±0.02	0.13±0.017 A
Rp5	0.16±0.01	0.15±0.011
Rp6	0.17±0	0.17±0.009
Rp9	0.12±0.01	0.12±0.013 A
UTC	0.12±0.01 A	0.11±0.009 A

UTC- untreated control;   Means are represented as average of three replicates ­± S.D

Means not labelled with the superscript letter A are significantly different from the control level mean according to Dunnet  Multiple Comparison  Test at P = 0.05.

In our studies wheat seedlings treated with IAA-producing bacterial isolates Et1, Rp1, Rp5, Rp6, and Rp9 showed striking shift in the root system architecture, demonstrating significant expansion of lateral roots and root hairs in comparison to untreated control(Fig 5b).Several workers have previously described similar findings caused by inoculation with  Bacillus altitudinis (strain FD48) in rice³¹, Phyllobacterium brassicacearum STM196 strain in Arabidopsis thaliana³². Auxins and cytokinins  plays significant role in lateral root and root hair development^32,33.

Figure 5: a) Effect of bio inoculation treatment on wheat seedlings with UTC ( untreated control);b) Root morphology of PGPR treated and UTC (untreated  control) on wheat seedlings Click here to view Figure

According to Ambreetha et al.(2018)²⁷ and López-Bucio et al.(2007)³⁴, accumulation of IAA in the rice plant’s root system after treatment with Bacillus increases the number of roots and lateral roots, thickness, area, and volume of the roots, compared to untreated plant similar results were obtained by Patten & Glick(2002)²⁵ in the treatment of mung bean seeds with Pseudomonas putida GR12-2.

Conclusion

The current investigation’s findings highlight the significance of the rhizosphere of the native plant Pterocarpus marsupium Roxb. as a source of IAA producing PGPR. Among five isolate Rp6 appears to be more promising as it produces high amount of IAA at diverse culture condition ( pH, temperature, incubation period etc.) hence it can enhance plant growth at diverse climatic conditions in the field. All the isolates under study enhanced wheat growth and development of lateral roots and root hairs. As a result, additional research is required to determine whether any of the chosen isolates have the potential to be a viable biofertilizer in the field settings. Therefore, it can be concluded that IAA producing isolates obtained from rhizosphere of Pterocarpus marsupium Roxb. species have a significant impact on plant growth and development and have prospective as a biofertilizer for sustainable agriculture.

Acknowledgement

 The Authors are grateful to Dr. Sanjay Kharat, Principal, Modern College, Ganeshkhind, Pune and Dr. Vinay Kumar, Head, Department of Biotechnology and Dr. Rekha Gupta, former Head Department of Biotechnology, Principal B.G. College, Sangvi, Principal, T. C. College, Baramati for facilities and support.

Funding Sources

There are no funding sources.

Conflict of Interest

There is no conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Conflict of Interest 

Authors declare that they have no conflict of interest.

Ethical Approval Statement

This article does not contain any studies with human participants or animals.

Author’s Contribution

Conceptualization and designing of the research work (VR, RB,SA ); Execution of lab experiments and data collection (VR ); Analysis of data and interpretation (VR ); Preparation of manuscript (VR,RB ).

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